Fluorescent polarization immunoassay utilizing substituted triazinylaminofluorescein aminoglycosides

ABSTRACT

This disclosure relates to a method and reagents for determining ligands in biological fluids such as serum, plasma, spinal fluid, amnionic fluid and urine. In particular, this disclosure relates to a fluorescent polarization immunoassay procedure and to a novel class of tracer compounds employed as reagents in such procedures. The procedure disclosed combines the specificity of an immunoassay with the speed and convenience of fluorescent polarization techniques to provide a means for determining the amount of a specific ligand present in a sample.

This is a division of application Ser. No. 325,872, filed Nov. 30, 1981,now U.S. Pat. No. 4,420,568, which is a continuation-in-part ofapplication Ser. No. 173,553, filed July 30, 1980, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a method and reagents for determiningligands in biological fluids such as serum, plasma, spinal fluid,amnionic fluid and urine. In particular, the present invention relatesto a fluorescent polarization immunoassay procedure and to tracersemployed as reagents in such procedures. The fluorescent polarizationimmunoassay procedure of the present invention combines the specificityof an immunoassay with the speed and convenience of fluorescentpolarization techniques to provide a means for determining the amount ofa specific ligand present in a sample.

Competitive binding immunoassays for measuring ligands are based on thecompetition between a ligand in a test sample and a labeled reagent,referred to as a tracer, for a limited number of receptor binding siteson antibodies specific to the ligand and tracer. The concentration ofligand in the sample determines the amount of tracer that willspecifically bind to an antibody. The amount of tracer-antibodyconjugate produced may be quantitively measured and is inverselyproportional to the quantity of ligand in the test sample.

In general, fluorescent polarization techniques are based on theprinciple that a fluorescent labeled compound when excited by linearlypolarized light will emit fluorescence having a degree of polarizationinversely related to its rate of rotation. Therefore, when a moleculesuch as a tracer-antibody conjugate having a fluorescent label isexcited with linearly polarized light, the emitted light remains highlypolarized because the fluorophore is constrained from rotating betweenthe time light is absorbed and emitted. When a "free" tracer compound(i.e., unbound to an antibody) is excited by linearly polarized light,its rotation is much faster than the corresponding tracer-antibodyconjugate and the molecules are more randomly oriented, therefore, theemitted light is depolarized. Thus, fluorescence polarization provides aquantitive means for measuring the amount of tracer-antibody conjugateproduced in a competitive binding immunoassay.

Various fluorescent labeled compounds are known in the art. U.S. Pat.No. 3,998,943 describes the preparation of a fluorescently labeledinsulin derivative using fluorescein isothiocyanate (FITC) as thefluorescent label and a fluorescently labeled morphine derivative using4-aminofluorescein hydrochloride as the fluorescent label. Blakeslee, etal, in The Journal of Immunological Methods, 13, 305-320 (1976)described the 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein derivatiesof immunoglobulins (IgG) having molecular weights of at least 160,000.Blakeslee fails to teach or suggest the use of the various fluoresceinderivatives in conjunction with fluorescent polarization immunoassaytechniques.

SUMMARY OF THE INVENTION

The present invention encompasses a method for determining ligands in asample comprising intermixing with said sample a biologically acceptablesalt of a tracer of the formula: ##STR1## wherein Y is halo or loweralkyl; and

R is a ligand-analog wherein said ligand-analog has at least one commonepitope with said ligand so as to be specifically reconizable by acommon antibody;

and an antibody capable of specifically recognizing said ligand and saidtracer; and then determining the amount of tracer-antibody conjugate byfluorescence polarization techniques as a measure of the concentrationof said ligand in the sample.

The invention further relates to a novel class of tracers of formula (I)and biologically acceptable salts thereof, which are useful in reagentsin the above-described method. The methods and tracers of the presentinvention are particularly useful in quantitatively monitoringtherapeutic drug concentrations in serum and plasma.

DETAILED DESCRIPTION OF THE INVENTION

The term "ligand" as used herein refers to a molecule in particular alow molecular weight hapten, to which a receptor, normally an antibody,can be obtained or formed. Haptens are protein-free bodies, generally oflow molecular weight that do not induce antibody formation when injectedinto an animal, but are reactive to antibodies. Antibodies to hapten aregenerally raised by first conjugating the haptens to a protein andinjecting the conjugate product into an animal. The resulting antibodiesare isolated by conventional antibody isolation techniques.

The ligands determinable by the method of the present invention varyover a wide molecular weight range. Although high molecular weightligands may be determined, for best results, it is generally preferableto employ the methods of the present invention to determine ligands oflow molecular weight, generally in a range of 50 to 4000. It is morepreferred to determine ligands having a molecular weight in a range of100 to 2000.

The novel tracer of the present invention includes compounds of formula(I) wherein the ligand-analogs represented by R include radicals havinga molecular weight within a range of 50 to 4000. The preferred noveltracers include compounds of formula (I) wherein the ligand-analogsrepresented by R include radicals having a molecular weight within arange of 100 to 2000.

Representative of ligands determinable by the methods of the presentinvention include steroids such as estrone, estradiol, cortisol,testosterone, progesterone, chenodeoxycholic acid, digoxin, cholic acid,digitoxin, deoxycholic acid, lithocholic acids and the ester and amidederivatives thereof; vitamins such as B-12, folic acid, thyroxine,triiodothyronine, histamine, serotorin, prostaglandins such as PGE, PGF,PGA; antiasthamatic drugs such as theophylline, antineoplastic drugssuch as doxorubicin and methotrexate; antiarrhythmic drugs such asdisopyramide, lidocaine, procainamide, propranolol, quinidine,N-acetylprocainamide; anticonvulsant drugs such as phenobarbital,phenytoin, primidone, valproic acid, carbamazepine and ethosuximide;antibiotics such as penicillins, cephalosporins, erythromycin,vancomycin, gentamicin, amikacin, chloramphenicol, streptomycin andtobramycin; antiarthritic drugs such as salicylate; antidepressant drugsincluding tricyclics such as nortrptyline, amitriptyline, imipramine anddesipramine; and the like as well as the metabolites thereof.

Additional ligands that may be determined by the methods of the presentinvention include drugs of abuse such as morphine, heroin, hydromophone,oxymorphone, metapon, codeine, hydrocodone, dihydrocodiene,dihydrohydroxy codeinone, pholcodine, dextromethorphan, phenazocine anddeonin and their metabolities.

The tracers of the present invention generally exist in an equilibriumbetween their acid and ionized states, and in the ionized state areeffective in the method of the present invention. Therefore, the presentinvention comprises the tracers in either the acid or ionized state andfor convenience, the tracers of the present invention are structurallyrepresented herein in their acid form. When the tracers of the presentinvention are present in their ionized state, the tracers exist in theform of biologically acceptable salts. As used herein, the term"biologically acceptable salts" refers to salts such as sodium,potassium, ammonium and the like which will enable the tracers of thepresent invention to exist in their ionized state when employed in themethod of the present invention. Generally, the tracers of the presentinvention exist in solution as salts, the specific salt results from thebuffer employed, i.e., in the presence of a sodium phosphate buffer, thetracers of the present invention will generally exist in their ionizedstate as a sodium salt.

The tracers of the present invention comprise a ligand-analogrepresented by R linked to a triazinylaminofluorescein moiety of theformula: ##STR2## wherein Y is above defined. Representation of the"lower alkyl" groups represented by Y include alkyl radicals having from1 to 4 carbon atoms, such as, for example, methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, t-butyl, and the like. Illustrative ofthe "halo" groups represented by Y include fluoro, chloro, iodo andbromo. It is preferred that Y is chloro or bromo and most preferred thatY is chloro.

The term ligand-analog as used herein refers to a mono- or polyvalentradical a substantial proportion of which has the same spatial and polarorganization as the ligand to define one or more determinant or epitopicsites capable of competing with the ligand for the binding sites of areceptor. A characteristic of such ligand-analog is that is possessessufficient structural similarity to the ligand of interest so as to berecognized by the anitbody for the ligand. For the most part, the ligandanalog will have the same or substantially the same structure and chargedistribution (spatial and polar organization) as the ligand of interestfor a significant portion of the molecular surface. Since frequently,the linking site for a hapten will be same in preparing the antigen forproduction of antibodies as used for linking to the ligand, the sameportion of the ligand analog which provides the template for theantibody will be exposed by the ligand analog in the tracer.

In general, the class of ligand analogs represented by R are derivedfrom the corresponding ligand by removal of a reactive hydrogen atom,i.e., a hydrogen atom bonded to a hydroxy oxygen or a reactive amine(primary or secondary) or by the formation of an amino derivative of theligand wherein an imino group ##STR3## replaces one or more atomsoriginally present in the ligand, at the site of binding to thetriazinylaminofluorescein moiety. Illustrative of ligands which upon theremoval of a reactive hydrogen may form a ligand-analogs represented byR include for example, procainamide, thyroxine, quinidine and theaminoglycoside antibiotics. Illustrative of ligands whose aminoderivatives are useful as ligand-analog include theophylline, valproicacid, phentobarbital, phenytoin, primidone, disopyramide, digoxin,chloramphenicol, salicylate, acetaminophen, carbamazepine, desimpramineand nortriptyline. In addition, a ligand may be structurally modified bythe addition or deletion of one or more functional groups to form aligand-analog, while retaining the necessary epitope sites for bindingto an antibody. However, it is preferred that such modifiedligand-analogs be bonded to the triazinylaminofluorescein moeity throughan imino or oxy group.

The tracers of the present invention are generally prepared inaccordance with the following procedure:

    R--X                                                       (III)

wherein R is above-defined and X is a reactive hydrogen; is reacted witha compound of the formula: ##STR4## wherein Z is halo and Y isabove-defined, and wherein the amino group is bonded to the 4 or 5position of the benzoic acid ring; under basic conditions in thepresence of an intert solvent to yield a compound of formula (I).

It should be noted that if a compound of formula (III) has more than onereactive hydrogen, a mixture of products of formula (I) may result uponreaction with a triazinylaminofluorescein moiety. For example, when theligand of interest is an antibiotic such as, for example, anaminoglycoside, the corresponding ligand-analog is generally derivedfrom a ligand having multiple reactive amine hydrogens. Replacing any ofsuch reactive amine hydrogens with a triazinylaminofluorescein moietywill produce a tracer of formula (I). Therefore, the reaction productresulting from the reaction of an antibiotic such as an aminoglycoside,with triazinylaminofluorescein will generally be a mixture of productsrepresented by formula (I). All of these reaction products, individuallyor in combination are effective as tracers in a fluorescent polarizationimmunoassay technique.

The temperature at which the reaction for preparing the tracers of thisinvention proceeds is not critical. The temperature should be one whichis sufficient so as to initiate and maintain the reaction. Generally,for convenience and economy, room temperature is sufficient. Inpreparing the tracers of the present invention, the ratio of reactantsis not narrowly cirtical. For each mole of a compound of formula (II),one should employ one more of a compound of formula (III) to obtain areasonably yield. It is preferred to employ an excess of compound offormula (III) for ease of reaction and recovery of the reactionproducts.

The compounds of formula (IV) employed as starting materials in theproduction of the tracers of this invention are prepared in accordancewith the method described by Blakeslee, et al. (supra). It should benoted that two isomers of compound (IV) generally exist. Isomer I isprepared from 5-aminofluorescein and Isomer II is prepared from4-amino-fluorescein. It is preferred to employ Isomer I or Isomer II andmixtures thereof as starting materials in the preparation of thecompounds of the present invention.

For ease of handling and recovery of product, the process for preparingthe tracers of the present invention is conducted in the presence of aninert solvent. Suitable inert solvents include those solvents which donot react with the starting materials and are sufficient to dissolve thestarting materials, and include for example water, methanol,dimethylformamide, dimethylsulfoxide and the like. In order to providemaximum product yields, the reaction preferably proceeds under neutralor basic conditions. If the compound of formula (III) is a reactiveamine salt, a suitable base is added to the reaction mixture to form thefree base of the reactive amine. Suitable bases include for exampletriethylamine. The reaction products of formula (I) are generallypurified using either thin-layer or column chromatography prior toapplication in the methods of the present invention.

In accordance with the method of the present invention, a samplecontaining the ligand to be determined is intermixed with a biologicallyacceptable salt of a tracer of formula (I) and an antibody specific forthe ligand and tracer. The ligand present in the sample and the tracercompete for limiting antibody sites resulting in the formation ofligand-antibody and tracer-antibody complexes. By maintaining constantthe concentration of tracer and antibody, the ratio of ligand-antibodycomplex to tracer-antibody complex that is formed is directlyproportional to the amount of ligand present in the sample. Therefore,upon exciting the mixture with fluorescent light and measuring thepolarization of the fluorescence emitted by a tracer and atracer-antibody complex, one is able to quantitatively determine theamount of ligand in the sample.

In theory, the fluorescence polarization of a tracer not complexed to anantibody is low, approaching zero. Upon complexing with a specificantibody, the tracer-antibody complex thus formed assumes the rotationof the antibody molecule which is slower than that of the relativelysmall tracer molecule, thereby increasing the polarization observed.Therefore, when a ligand competes with the tracer for antibody sites,the observed polarization of fluorescence of the tracer-antibody complexbecomes a value somewhere between that of the tracer and tracer-antibodycomplex. If a sample contains a high concentration of a ligand, theobserved polarization value is closer to that of the free ligand, i.e.,low. If the test sample contains a low concentration of the ligand, thepolarization value is closer to that of the bound ligand, i.e., high. Bysequentially exciting the reaction mixture of an immunoassay withvertically and then horizontally polarized light and analyzing only thevertical component of the emitted light, the polarization offluorescence in the reaction mix may be accurately determined. Theprecise relationship between polarization and concentration of theligand to be determined is established by measuring the polarizationvalues of calibrators with known concentrations. The concentration ofthe ligand can be extrapolated from a standard curve prepared in thismanner.

The pH at which the method of the present invention is practiced must besufficient to allow the tracers of formula (I) to exist in their ionizedstate. The pH may range from about 3 to 12, more usually in the range offrom about 5 to 10, most preferably from about 6 to 9. Various buffersmay be used to achieve and maintain the pH during the assay procedure.Representative buffers include borate, phosphate, carbonate, tris,barbital and the like. The particular buffer employed is not critical tothe present invention, but in an individual assay, a specific buffer maybe preferred in view of the antibody employed and ligand to bedetermined. The cation portion of the buffer will generally determinethe cation portion of the tracer salt in solution.

The methods of the present invention are practical at moderatetemperatures and preferably at a constant temperature. The temperaturewill normally range from about 0° to 50° C., more usually from about 15°to 40° C.

The concentration of ligand which may be assayed will generally varyfrom about 10⁻² to 10⁻¹³ M, more usually from about 10⁻⁴ to 10⁻¹⁰ M.Higher concentrations of ligand may be assayed upon dilution of theoriginal sample.

In addition to the concentration range of ligand of interest,considerations such as whether the assay is qualitative,semiquantitative or quantitative, the equipment employed, and thecharacteristics of the tracer and antibody will normally determine theconcentration of the tracer and antibody to be employed. While theconcentration of ligand in the sample will determine the range ofconcentration of the other reagents, i.e., tracer and antibody, normallyto optimize the sensitivity of the assay, individual reagentconcentrations will be determined empirically. Concentrations of thetracer and antibody are readily ascertained by one of ordinary skill inthe art.

As previously mentioned the preferred tracers of the present inventionare prepared from 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein or4-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and exist preferably asisomers of the formula: ##STR5##

The following illustrative, nonlimiting examples will serve to furtherdemonstrate to those skilled in the art the manner in which specifictracers within the scope of this invention may be prepared. The symbol[DTAF] appearing in the structural formulas illustrating the compoundsprepared in the following examples, represents a moiety of the formula:##STR6## wherein the imino nitrogen is attached to the 4 to 5 positionin the above formula depending on the specific triazinylfluoresceinisomer employed as the starting material.

EXAMPLE I

Gentamicin sulfate (200 mg) was dissolved in 1 ml of distilled water andthe resulting solution was adjusted to pH 9.0 using approximately 0.8 mlof 1.0M sodium hydroxide.5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein (20 mg) was dissolved in1.5 ml of methanol and the resulting methanol solution was addeddropwise to the gentamicin solution with stirring. The reaction mixturewas allowed to react for one hour. The resultant mixture waschormatographed on a DEAE cellulose medium mesh column using 0.1Mphosphate buffer at pH 8.0 as the eluent to yield a gentamicin-DTAfconjugate.

EXAMPLE II

Tobramycin (250 mg) was dissolved in 2 ml of 0.1M carbonate buffer (pH9.0). 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein (20 mg) wasdissolved in 1 ml of methanol and the resulting methanol solution wasadded to 1 ml of the tobramycin solution. After approximately fiveminutes, the rection mixture was purified by chormatography on a DEAEcellulose column using 0.1M phosphate buffer at pH 8.0 as the eluent toyield a tobramycin-DTAF conjugate.

EXAMPLE III

Amikacin (9.24 mg) was dissolved in 0.2 ml of water. A suspensioncontaining 4.5 mg of dichlorotriazinyl-aminofluorescein in 0.2 ml ofmethanol was added to the amikacin solution with stirring. The smallparticles of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein rapidlydissolved, the reaction mixture was chromatographed on a 17 ml column ofDEAE cellulose using pH 8.1 phosphate buffer, 0.1M as the eluent toyield an amikacin-DTAF conjugate.

EXAMPLE IV

Streptomycin sulfate (200 mg) was dissolved in 15 ml of water and theresulting solution was adjusted to pH 10.5 using 1N sodium hydroxide. Tothe streptomycin solution was dropwise added with stirring, 20 mg of5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in 1.5 ml ofdimethylsulfoxide. 4 ml of the reaction mixture was chromatographed on aDEAE cellulose column using a 0.1M phosphate buffer (pH 8.0) as theeluent to yield a streptomycin-DTAF conjugate.

EXAMPLE V

Neomycin sulfate (200 mg) was dissolved in 3 ml of water and theresulting solution was adjusted to pH 9.0 using 6N sodium hydroxide. Tothe neomycin solution was dropwise added with stirring 20 mg of5-[(4,6-dichlorotriazin-2-yl)-amino]-fluorescein dissolved in 1.5 ml ofdimethylsulfoxide. 2 ml of the reaction mixture was chromatographed on aDEAE cellulose column using 0.1M phosphate buffer (pH 8.0) as the eluentto yield a neomycin-DTAF conjugate.

EXAMPLE VI

Vancomycin hydrochloride (100 mg) was dissolved in 100 ml of water andthe resulting solution was adjusted to pH 9.1 using 1N sodium hydroxide.The vancomycin solution was then adjusted to pH 7 using 1N HCl afterwhich time 20 mg of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein in 2ml of dimethylsulfoxide was added with stirring. A crude product formedwhich was purified using silica gel thin-layer chromatography techniquesemploying a developing solvent comprising a chloroform-methanol-water(4:4:1) mixture to yield a vancomycin-DTAF conjugate.

EXAMPLE VII

To 1.79 g of para-acetamidobenzoic acid and 1.15 g ofN-hydroxysuccinimide dissolved in 15 ml of pyridine was added 2.3 g ofN,N'-dicyclohexylcarbodiimide. The reaction mixture was cooled at 4° C.for two hours and then filtered to remove crystals which had formed. Thecrystals were washed with approximately 2 ml acetone and the pyridinefiltrate and acetone washings were then combined. To the combinedmixture was added 0.88 g of N-ethylethylenediamine. The resultingmixture was stirred for two hours and then cooled at 4° C. for abouttwenty-four hours to yield a second crop of crystals. The crystals wereremoved from the mixture by filtration and then rinsed with acetone. Thetwo crops of crystals (approximately 2.0 g) were combined and thendissolved in 50 ml of distilled water. The pH of the resulting mixturewas adjusted to pH 10 using a 6N sodium hydroxide solution. A whiteprecipitate, desethyl-N-acetylprocainamide formed and was removed byfiltration and dried in a dessicator. To 10 mg of thedesethyl-N-acetyl-procainamide was added 10 mg of4-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in 1 ml ofmethanol. The mixture was allowed to react for ten minutes, and thecrude product which had formed was purified using silica gel thin-layerchromatographic techniques employing a developing solvent comprising a1:1 mixture of chloroform: acetone to yield adesethyl-N-acetyl-procainamide-DTAF conjugate of the formula: ##STR7##

EXAMPLE VIII

The procedure of Example IV was employed using 0.9 g of ethylenediaminein lieu of N-ethylethylenediamine. The reaction mixture was stirred forone hour, and cooled for 1.5 hours. Methanol was used in lieu of a 1:1mixture of chloroform:acetone as the developing solvent in thepurification of the crude product to yield an N-p-acetamidobenzoylethylene diamine-DTAF conjugate of the formula: ##STR8##

EXAMPLE IX

A mixture containing desethyl-N-acetyl procainamide (1.25 g) (preparedas in Example VII and 0.8 g of chloroacetyl chloride dissolved in 25 mlof acetone was refluxed for two hours. The reaction mixture was filteredand the filtrate evaporated to yield a yellow residue. The yellowresidue and 0.75 g of sodium iodide were dissolved in 20 ml of acetoneand refluxed for one hour. The resulting mixture was filtered and thefiltrate evaporated to dryness to yield a red residue which was thendissolved in 20 ml of methanol. To the methanol solution was added 20 mlof concentrated ammonium hydroxide and the resulting mixture wasrefluxed for 1.5 hours. The mixture was cooled and then extracted twicewith 20 ml of chloroform. The combined extracts were dried over sodiumsulfate, filtered and evaporated to yieldN-p-acetamidobenzoyl-N'-ethyl-N'-aminoacetylethylene diamine. To 10 mgof N-p-acetamidobenzoyl-N'-ethyl-N'-aminoacetylethylene diamine wasadded 10 mg of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolvedin 1 ml of methanol and the reaction was allowed to proceed for tenminutes until a crude product had formed. The crude product was purifiedusing silica gel thin-layer chromatographic techniques employing adeveloping solvent comprising a 1:1 mixture of chloroform:acetone toyield an N-P-acetamidobenzoyl-N'-ethyl-N'-aminoacetylethylenediamine-DTAF conjugate of the formula: ##STR9##

EXAMPLE X

To 1.1 g of primidone dissolved in 10 ml of concentrated sulfuric acidwas slowly added a solution containing 1 ml of concentrated nitric acidand 2 ml of concentrated sulfuric acid. The reaction mixture was shakenat room temperature for forty-five minutes. The reaction mixture wasthen poured over 50 ml ice and crystals of para-nitroprimidone that hadformed were filtered and then rinsed with water. The crystals (1.17 g,having a melting point 225°-228° C.) were dissolved in 200 ml of hotethanol. To the ethanol solution was added 1.5 g of iron powder and 100ml of water. The resultant mixture was heated to boiling, and then 2 mlof concentrated hydrochloric acid was added. The resultant mixture wasrefluxed for two hours and the hot mixture was filtered and the filtrateto yield 0.8 g of brown hydroscopic crystals. 5 mg of5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and 5 mg of the browncrystals were dissolved in 0.5 ml of methanol. The reaction was completein 10 minutes to yield a crude product which was purified by silica gelthin-layer chromatography using 3:1 mixture of chloroform: methanol asthe developing solvent. The product was further purified usingthin-layer chromatography employing a 2:1 mixture of chloroform:methanolto yield an amino-primidone-DTAF conjugate of the formula: ##STR10##

EXAMPLE XI

To delta-valerolactam (10 g) dissolved in 80 ml of dry tetrahydrofuran,under a dry nitrogen atmosphere was deopwise added n-butyllithium (1.6M,125 ml) in hexane while the reaction mixture was chilled in a dryice-acetone bath. After all the n-butyllithium was added, the reactionmixture was stirred at room temperature for one hour, refluxed forthirty minutes, and cooled to room temperature. 1-Bromopropane (12.3 g)was slowly added to the reaction mixture while the mixture was chilledin an ice bath. The reaction mixture was then stirred for sixteen hoursat room temperature. 100 ml of water was added slowly to the reactionmixture and the resulting mixture was stirred at room temperature forthirty minutes. The layers separated and the aqueous layers werecombined, dried over sodium sulfate, and then evaporated to yield 12.4 g(88% yield) of a dark, heavy oil, which crystallized on standing. Thecrystals were recrystallized from petroleum ether to yield 5.4 g ofα-propylvalerolactam (m.p. 75°-76° C.). A portion of theα-propylvalerolactam (2.8 g) was refluxed in 25 ml of 6N hydrochloricacid under a nitrogen atmosphere for six hours. The water was evaporatedto yield 2-propyl-5-amino-pentanoic acid (2.0 g; 51% yield) andrecrystallized from ethanol-petroleum ether to yield a solid (m.p.71°-73° C.). Equimolar amounts of 2-propyl-5-amino-pentanoic acid and5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein were dissolved inmethanol. The reaction was completed in about ten minutes to yield acrude product which was purified by silica gel thin-layer chromatographywith chloroform/methanol (3:1) as the developing solvent to yield a2-propyl-5-amino-pentanoic acid-DTAF conjugate of the formula: ##STR11##

EXAMPLE XII

The procedure of Example VIII was employed utilizing2-ethyl-5-aminopentanoic acid in lieu of 2-propyl-5-amino-pentanoic acidto yield a 2-ethyl-5-amino-pentanoic acid-DTAF conjugate of the formula:##STR12##

EXAMPLE XIII

To a mixture containing 5 mg of5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in methanoland 5 mg of D-thyroxine was dropwise added dimethylsulfoxide until aclear solution was formed. Two drops of triethylamine was added to thereaction mixture and the reaction was allowed to proceed for 16 hours. Acrude product has formed which was then purified by silica gelthin-layer chromatography using a 3:1 mixture of chloroform:methanol asthe developing solvent to yield a 3,3',5,5'-tetraiodo-D-thyronine-DTAFconjugate of the formula: ##STR13##

EXAMPLE XIV

The procedure of Example XIII was employed utilizing L-thyroxine in lieuof D-thyroxine to yield 3,3',5,5'-tetraiodo-L-thyronine-DTAF conjugatewhich is an optical isomer of the conjugate formed in Example XIII.

EXAMPLE XV

The procedure of Example XIII was employed utilizing3,3',5-triiodo-L-thyronine in lieu of D-thyroxine to yield a3,3',5-triiodo-L-thyronine-DTAF conjugate of the formula: ##STR14##

EXAMPLE XVI

A solution containing ammonium acetate (8.0 g), sodium cyanoborohydride(630 mg) and 10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5-one (2.1 g)dissolved in 50 ml of methanol was refluxed for twenty four hours andthen evaporated to dryness to yield a tan residue. The residue wasdissolved in 25 ml of 2N hydrochloric acid and extracted twice with 25ml dichloromethane. 6N sodium hydroxide was added to the aqueous phaseuntil the pH of the solution was 14. A brown oil then began to form andthe solution was chilled in a freezer for 16 hours. All water in themixture was evaporated and the residue was taken up in methanol andfiltered. The filtrate was evaporated to yield a white residue. 10 mg of5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and 10 mg of the whiteresidue were dissolved in 1 ml of methanol to yield a crude productwhich was purified by silica gel thin-layer chromatography using a 1:1mixture of chloroform:acetone as a developing solvent to yield a5-amino-10,11-dihydro-5H-dibenzo[a,d]-cycloheptene-DTAF conjugate of theformula: ##STR15##

EXAMPLE XVII

A mixture containing 10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-one (10g) and dimethylhydrazine (18%) were refluxed for twenty-four hours in100% ethanol. To the mixture was added 100 ml of distilled water and ayellow solution was extracted with diethyl ether until the extracts werecolorless. The combined ether extracts were washed with 25 ml of 2Nhydrochloric acid. The organic phase was then dried over sodium sulfateand evaporated to yield dibenzosuberone dimethyl hydrazone as a thickorange oil. This oil (2.0 g) was refluxed for twelve hours in a solutioncontaining 3 g of hydrazine in 10 ml of 100% ethanol. The reactionmixture was poured over 10 ml of ice water then extracted twice with 25ml of diethyl ether. The combined ether extracts were dried over sodiumsulfate and evaporated to dryness to yield dibenzosuberone hydrazone asa yellow oil. 10 mg of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluoresceinand 10 mg of dibenzosuberone hydrazone in 1 ml of methanol were allowedto react for 10 minutes to yield a crude product which was purifiedusing silica gel thin-layer chromatography using a 3:1 mixture ofchloroform:methanol as a developing solvent to yield a dibenzosuberonehydrazone-DTAF conjugate of the formula: ##STR16##

EXAMPLE XVIII

5-(γ-Bromopropylidene)-5H-dibenzo[a,d]-10,11-dihydro cycloheptene andits precursor5-cyclopropyl-5-hydroxy-5H-dibenzo[a,d]-10,11-dihydrocycloheptene wereprepared by the procedure described in The Journal of Organic Chemistry,Vol. 27, pages 4134-4137 (1962) by R. D. Hoffsomer, D. Taub, and N. L.Wendler. Procedure (b) for preparation of end product,5-(γ-aminopropylidene)-5H-dibenzo[a,d]-10,11-dihydrocycloheptene, wasemployed substituting the bromopropylidene compound for thechloropropylidene compound. 10 mg of5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and 10 mg of the endproduct were dissolved in 1 ml of methanol. An excess amount oftriethylamine was added and the reaction was completed in thirty minutesto yield a crude product which was purified by silica gel thin-layerchromatography using a 2:1 mixture of chloroform:methanol as developingsolvent to yield a5-(γ-aminopropylidene)-5H-dibenzo[a,d]-10,11-dihydrocycloheptene-DTAFconjugate of the formula: ##STR17##

EXAMPLE XIX

To a mixture containing 6.0 g of iminodibenzyl in 30 ml of chloroformwas added 6 ml of chloroacetly chloride and the resultant mixture wasrefluxed for forty-five minutes. To the reaction mixture was added 60 mlof water and the resultant mixture was stirred for thirty minutes atroom temperature. The chloroform layer was separated and dried oversodium sulfate and evaporated to dryness to yield a residue. The residuewas dissolved in 25 ml of acetone. A solution containing 4.5 g sodiumiodide dissolved in 25 ml of acetone was added to the acetone solutionand the resultant mixture was refluxed for thirty minutes. To thereaction mixture was added 100 ml of water and the reaction mixture wasextracted twice with 50 ml of chloroform and evaporated to dryness toyield a residue which was then dissolved in 40 ml of methanol. To themethanol solution was added 60 ml of concentrated ammonium hydroxide andthe resultant mixture was refluxed for one hour. The reaction mixturewas evaporated to dryness and the residue was taken up in 100 ml ofchloroform and washed twice with 30 ml of water. The chloroform layerwas dried over sodium sulfate and evaporated to dryness to yield 4.5 gof an amine product. A mixture containing 5 mg of5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and 5 mg of the aminedissolved in 0.5 ml of methanol were allowed to react for 10 minutes toyield a crude product which was purified as in Example XVI to yield aN-aminoacetyliminostibene-DTAF conjugate of the formula: ##STR18##

EXAMPLE XX

A mixture containing desipramine hydrochloride (1.33 g) and chloroacetylchloride (0.8 g) dissolved in 25 ml chloroform was refluxed for twohours. The chloroform was evaporated to yield a residue which wasdissolved in 25 ml of acetone. Sodium iodide (0.75 g) was added to theacetone solution and the resultant solution was refluxed for thirtyminutes and then filtered. The precipitated salt was rinsed with acetoneand the acetone filtrate was evaporated and the residue was taken up in20 ml of methanol. To the methanol solution was added 20 ml ofconcentrated ammonium hydroxide and the resultant solution was refluxedfor one hour. The reaction mixture was extracted three times with 25 mlof chloroform and combined extracts were dried over sodium sulfate,filtered and evaporated. 5 mg of5-[(4,6-dichlorotriazin-2-yl)amino]fluorescein and 5 mg of the aminewere dissolved in 0.5 ml of methanol. About five drops ofdimethylsulfoxide were added to the reaction mixture to dissolve theprecipitate. The reaction was completed in ten minutes and yielded acrude product which was purified in accordance with the procedure ofExample XVI using a 3:1 mixture of chloroform:methanol as the developingsolvent to yield a N-aminoacetyldesipramine-DTAF conjugate of theformula: ##STR19##

EXAMPLE XXI

5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein (10 mg) and8-aminomethyl-theophylline (5 mg) were dissolved in 0.5 ml ofdimethylsulfoxide. After five minutes the reaction was complete andyielded a crude product which was purified by silica gel thin-layerchromatography using a 1:1 mixture of chloroform:acetone as thedeveloping solvent to yield an 8-aminomethyl-theophylline-DTAF conjugateof the formula: ##STR20##

EXAMPLE XXII

The procedure of Example XXI was employed utilizing8-aminoethyl-theophylline in lieu of 8-aminomethyl-theophylline to yieldan 8-aminoethyl-theophylline-DTAF conjugate of the formula: ##STR21##

EXAMPLE XXIII

5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein (5 mg) and anhydrousquinidine (5 mg) were dissolved in 0.5 ml of dimethylformamide. Aftersixteen hours the reaction was complete and yielded a crude productwhich was purified by silica gel thin-layer chromatography, using a 3:1mixture of chloroform:methanol as the developing solvent to yield aquinidine-DTAF conjugate of the formula: ##STR22##

The following tracers were also prepared in accordance with the aboveprocedures:

EXAMPLE XXIV 5-(p-aminobenzamido)-2-propylpentanoic acid-DTAF conjugate##STR23## EXAMPLE XXV1-amino-2-phenyl-2-(2'-pyridyl)-4-diisopropylaminobutane-DTAF conjugate##STR24## EXAMPLE XXVI2-phenyl-2-(2'-pyridyl)-4-(diisopropylamino)-butyrylhydrazine-DTAFconjugate ##STR25## EXAMPLE XXVII 4-aminosalicyclic acid-DTAF conjugate##STR26## EXAMPLE XXVIII procainamide-DTAF conjugate ##STR27## EXAMPLEXXIX 1-hydroxymethyl-2-hydroxy-2-(4'-nitrophenyl)-ethylamine-DTAFconjugate ##STR28## EXAMPLE XXX 2-β-aminoethylphenytoin-DTAF conjugate##STR29## EXAMPLE XXXI 5-aminosalicyclic acid-DTAF conjugate ##STR30##EXAMPLE XXXII propanolol-DTAF conjugate ##STR31## EXAMPLE XXXIIIaminophenobarbital-DTAF conjugate ##STR32## EXAMPLE XXXIVα-aminoacetyliminostilbene-DTAF conjugate ##STR33## EXAMPLE XXXV1-N-isopropylamino-2-α-aminoacetyl-3-(1'-naphthoxy)-propane-[DTAF]conjugate ##STR34## EXAMPLE XXXVI 3-dehydroxy-3-aminodigoxigenin-DTAFconjugate ##STR35##

As mentioned above, the fluorescently labeled tracers prepared accordingto this invention can be used in a variety of immunoassay procedures inparticular in a fluorescence polarization immunoassay. The followingexamples demonstrate the suitability of tracers of the present inventionin assays employing fluorescence polarization techniques.

All examples followed the same general procedure:

(1) A small volume of standard or test serum is delivered into a testtube and diluted with buffer;

(2) A small volume of concentrated fluorescent tracer optionallycontaining a surfactant is then added to each tube;

(3) Finally, a volume of diluted antisera is added; and

(4) The reaction mixture is incubated at room temperature.

VALPROIC ACID ASSAY 2-ETHYL-5-AMINO-PENTANOIC ACID DTAF CONJUGATE

Materials Required:

(1) Buffer: 0.1M phosphate, pH 7.5, containing 0.01% (w/v) sodium azideand 0.01% (w/v) bovine gamma globulin (BGG).

(2) Tracer: 2-ethyl-5-amino-pentanoic acid-DTAF conjugate 50×10⁻⁹ M in0.1M tris hydrochloride buffer, pH 7.8, containing 0.1% (w/v) sodiumdodecyl sulfate, 0.01% (w/v) bovine gamma globulin, and 0.01% (w/v)sodium azide.

(3) Antibody: Sheep antiserum to valproic acid diluted to 1 to 3.75 inbuffer.

(4) Standards of unknowns: human serum (or other biological fluid)containing valproic acid in the concentration range 0 to 150 μg/ml.

(5) Fluorescence polarimeter: Instrument capable of reading thepolarization of fluorescence of a 1×10⁻⁹ M fluorescein solution to±0.001 polarization unit.

Protocol:

(1) 0.75 μl of standard or unknown sample placed in a 12×75 mmdisposable culture tube (cuvette). This is accomplished by pipetting 20μl of standard or unknown into a predilution container followed by 500μl of buffer. Next 20 μl of diluted sample is pipetted into the 12×75culture tube followed by 400 μl of buffer.

(2) 40 μl of tracer and 800 μl of buffer are added to the cuvette.

(3) 40 μl of antiserum and 800 μl of buffer are added to the cuvette.The contents of the cuvette are mixed and incubated for approximately 15minutes at room temperature.

(4) The fluorescence polarization is read. Typical results are presentedin Table I.

                  TABLE I                                                         ______________________________________                                        Valproic Acid Conc. (μg/ml)                                                                    Polarization                                              ______________________________________                                        0                   0.217                                                     12.5                0.186                                                     25                  0.165                                                     50                  0.132                                                     100                 0.099                                                     150                 0.081                                                     ______________________________________                                    

The polarization changes in a regular manner as the concentration ofvalproic acid is varied allowing the construction of a standard curve.Unknown samples are treated in an identical manner; from thepolarization of fluorescence of the unknown sample, the concentration ofvalproic acid in the unknown sample may be determined by reference tothe standard curve.

GENTAMICIN ASSAY

Materials Required:

(1) Buffer: (See valproic acid assay).

(2) Tracer: Gentamicin-DTAF at 100 nM in a trishydrochloride buffer pH7.5 containing 0.125% sodium dodecyl sulfate, 0.01% sodium azide, and0.01% bovine gamma globulin.

(3) Antibody: Rabbit or sheep antisera to gentamicin dilutedappropriately in buffer.

(4) Standards or unknowns: human serum (or other biological fluid)containing gentamicin.

(5) Fluorescence polarimeter: (See valproic acid assay).

Protocol:

(1) 1.8 μl of standard or unknown sample is placed in a 12×75 mmdisposable culture tube (cuvette). This is done by pipetting 20 μl ofsample followed by 200 μl of buffer. Next 20 μl of diluted sample ispipetted into the cuvette followed by 200 μl of buffer.

(2) 40 μl of tracer and 1000 μl of buffer are added to the cuvette.

(3) 40 μl of antibody and 1000 μl of buffer are added, the contents ofthe cuvette are mixed and incubated for approximately fifteen minutes atroom temperature.

(4) The fluorescence polarization is read following the incubation.Typical results are presented in Table II.

                  TABLE II                                                        ______________________________________                                        Gentamicin Concentration (μg/ml)                                                                Polarization                                             ______________________________________                                        0                    0.178                                                    0.5                  0.158                                                    1.0                  0.140                                                    2.0                  0.115                                                    4.0                  0.090                                                    8.0                  0.074                                                    ______________________________________                                    

The polarization changes in a regular manner allowing construction of astandard curve. Unknown samples are tested in an identical manner, andthe gentamicin content is determined by reference to the standard curve.The utility of the gentamicin-DTAF tracer for determining theconcentration of gentamicin in biological samples is therebyillustrated.

N-ACETYL PROCAINAMIDE ASSAY

Materials required:

(1) Buffer: (See valproic acid assay)

(2) Tracer: Desethyl-N-acetyl procainamide-DTAF conjugate at aconcentration of 50×10⁻⁹ in a 5.75% (w/v) solution of sodium toluenesulfonate.

(3) Antiserum: Rabbit antiserum to N-acetyl-procainamide diluted one tosix in buffer.

(4) Standards or unknowns: human serum (or other biological fluid).

(5) Fluorescence polarimeter: (See valproic acid assay).

Protocol:

(1) 0.48 μl of standard or unknown is placed in a cuvette by pipetting10 μl of sample into a predilution container and mixing with 200 μl ofbuffer. Ten μl of diluted sample is next pipetted into the cuvettefollowed by 200 μl of buffer.

(2) 40 μl of tracer and 1000 μl of buffer are added to the cuvette.

(3) 40 μl of antiserum and 1000 μl of buffer are next added to thecuvette. The contents of the cuvette are mixed and incubated at roomtemperature for approximately fifteen minutes at room temperature.

(4) The fluorescence polarization is read following the fifteen minuteincubation period. Typical results for the N-acetyl-procainamide arepresented in Table III.

                  TABLE III                                                       ______________________________________                                        N--Acetyl Procainamide (μg/ml)                                                                  Polarization                                             ______________________________________                                        0                    0.239                                                    1                    0.218                                                    2                    0.209                                                    4                    0.190                                                    8                    0.173                                                    16                   0.158                                                    ______________________________________                                    

The standard curve can be constructed from the data in Table III.Unknown samples treated identically to the standards can be quantitatedby references to the standard curve, thereby illustrating theusefullness of the standard N-acetyl procainamide-DTAF conjugate for thedetermination of N-acetyl-procainamide in biological fluids.

The following table summarizes the various fluorescence polarizationassays that have been carried out in accordance with the above-describedprocedures employing tracers prepared in the preceding examples. Thetracers employed are identified by Example number and the specificligand(s) determined are indicated.

    ______________________________________                                        Tracer Prepared                                                               In Example Number                                                                              Ligand(s) Assayed                                            ______________________________________                                        I                Gentamicin                                                   II               Tobramycin                                                   III              Amikacin                                                     IV               Streptomycin                                                 V                Neomycin                                                     VI               Vancomycin                                                   VII              N--acetylprocainamide                                        VIII             N--acetylprocainamide                                        IX               N--acetylprocainamide                                        X                Primidone                                                    XI               Valproic acid                                                XII              Valproic acid                                                XIII             Thyroxine (T.sub.4)                                          XIV              Thyroxine (T.sub.4)                                          XV               Thyroxine (T.sub.3)                                          XVI              Nortrptyline; Amitriptyline                                  XVII             Nortrptyline; Amitriptyline                                  XVIII            Nortrptyline; Amitriptyline                                  XIX              Imipramine; Desipramine                                      XX               Imipramine; Desipramine                                      XXI              Theophylline                                                 XXII             Theophylline                                                 XXIII            Quinidine                                                    XXIV             Valproic acid                                                XXV              Disopyramide                                                 XXVI             Disopyramide                                                 XXVII            Salicylate                                                   XXVIII           Procainamide                                                 XXIX             Chloroamphenicol                                             XXX              Phenytoin                                                    XXXI             Salicylate                                                   XXXII            Propranolol                                                  XXXIII           Phenobarbital                                                XXXIV            Carbamazepine                                                XXXV             Propranolol                                                  XXXVI            Digoxin                                                      ______________________________________                                    

As evident from the above results, the tracers of the present inventionare effective reagents in fluoroescence polarization immunoassays. Inaddition to the properties mentioned above, the tracers of the presentinvention posses a high degree of thermal stability, a high degree ofbound polarization, high quantum yields and are relatively easy toproduce and purify.

Although this invention has been described with respect to specificmodifications, the details thereof are not to be construed aslimitations, for it will be apparent that various equivalents, changesand modifications may be restorted to without departing from the spiritand scope thereof and it is understood that such equivalent embodimentsare intended to be included therein.

What is claimed is:
 1. A compound useful in a fluorescence polarizationimmunoassay for determining aminoglycosides, which compound has theformula: ##STR36## wherein Y is halo or lower alkyl; andR is aligand-analog having a molecular weight within the range of 50 to 4000wherein said ligand-analog has at least one common epitope with anaminoglycoside ligand so as to be specifically recognizable by a commonantibody;and biologically acceptable salts thereof.
 2. A compound ofclaim 1 wherein Y is halo.
 3. A compound of claim 2 wherein Y is chloro.4. A compound according to claim 1 wherein R is gentamicin.
 5. Acompound according to claim 1 wherein R is tobramycin.
 6. A compoundaccording to claim 1 wherein R is amikacin.
 7. A compound according toclaim 1 wherein R is streptomycin.